1. Field of the Invention
The invention relates to methods of detecting chemical ligation. One example application involves methods of detecting nucleic acids and, more specifically, to methods for the detection of ligation of nucleic acids via a change in fluorescence properties. Methods for the detection of ligation by the use of a quencher as a leaving group are disclosed.
2. Description of the Related Art
As the molecular nature of diseases is studied, there is an increasing need for methods of detecting and analyzing nucleic acids both in vitro and in vivo. Recent work on sequencing the human genome has resulted in a flood of genomic and proteomic information. This information has made a significant and sometimes life-determining difference in the diagnosis, prognosis, and treatment of disease. In order to fully take advantage of this information, more quick and simple, yet accurate, methods of detecting and analyzing the presence or absence of nucleic acids, which may differ by as little as one nucleotide from others, need to be developed. In certain cases, the nucleic acids may be present in minute quantities or concentrations, which underscores the need for high sensitivity as well.
For example, drug resistance in bacterial infections is typically characterized genetically. Methods for characterizing infections commonly involve first culturing the organism, which takes days at least and months at worst. A specific example is the standard diagnosis of tuberculosis, which commonly takes several weeks, as the Mycobacterium tuberculosis organism is slow growing, and determination of antibiotic resistance takes more time still. Even short (e.g. two days) bacterial cultures are dangerously long for patients with other acute infections such as those occurring in sepsis or in necrotizing fasciitis. Thus, methods for genetic analysis are increasingly important and faster methods are needed.
A standard and commonly used method of detecting target nucleic acids involves the use of oligonucleotides as hybridization probes in the field of chemistry, molecular biology and biotechnology. Oligonucleotide probes are synthesized to have sequences that are complementary to the target DNA or RNA strands, enabling the probes to hybridize to the target DNA or RNA strands under suitably stringent conditions. The standard procedure requires the DNA or RNA target strands to be immobilized on a solid surface, membrane, or bead. Then an oligonucleotide probe, labeled with a reporter group for identification, is added and binds non-covalently to any region of the target DNA or RNA strand encoding a complementary sequence to that of the probe. Next, any residual, unbound oligonucleotide probe is washed away from the immobilized target oligonucleotide, and the presence of any bound probe is detected by means of an attached reporter group. Common reporter groups include radioactive atoms (phosphorus, iodine, sulfur, carbon, or tritium), fluorescent or chemiluminescent groups, and enzymes that generate colored or fluorescent products. Many variations on this procedure exist which are known to those skilled in the art, including use of sandwich hybridization complexes, and in situ hybridization methodologies.
One limitation in using the standard hybridization method for detecting target nucleic acids is non-specific binding of the oligonucleotide probes to the target DNA or RNA. Short oligonucleotides (e.g., 6-12 mers) are much more effective at detecting single nucleotide mismatches than longer ones, but have a lower affinity to the template than longer oligonucleotides. Gryaznov and Letsinger developed a method of increasing the selectivity of the nucleic acid probe to the target nucleic acids, by using two or more shorter oligonucleotide probes instead of a single, long oligonucleotide probe. (Gryaznov, et al. Nucleic Acids Research, 22: 2366-2369, 1994; Letsinger et al., U.S. Pat. No. 5,681,943) The two or more shorter oligonucleotide probes would have either an electrophilic group (for example, bromoacetido, tosyl) or a nucleophilic group (for example, phosphorothioate monoester) at their termini. These shorter oligonucleotide probes contain base sequences that would bind to adjacent positions on a complementary template. When the probes align along the template, the oligonucleotide probes are brought into proximity of one another and spontaneously ligate and form an irreversible covalent bond. The oligonucleotide probes spontaneously ligate without any additional activating agents or enzymes.
Despite this improvement to the standard hybridization method, false positives may still result from the oligonucleotide probes non-selectively binding to proteins or the solid support. Standard hybridization methods using static labeling groups are further limited in that they usually have to be performed on solid supports under stringent conditions and require careful washing (static labeling here refers to labels, such as fluorescent labels, that do not change their signal). In particular, when standard oligonucleotide probes are used to detect or image nucleic acids in fixed cells, the cells have to be carefully prepared and the conditions properly manipulated to avoid nonspecific signals. Typically, cells are first fixed, permeabilized and crosslinked with formaldehyde and/or ethanol using procedures that are known to those skilled in the art. Next, hybridization is carried out, followed by several careful washes to remove unbound probes. Thus, standard hybridization methods using statically labeled oligonucleotides require time for preparation of the cells, increase the likelihood of error, and cannot be used in live cells, where washing away unbound probes is not possible.
In recent years, new methods for detecting nucleic acids that involve a change in fluorescence intensity or emission wavelength have been developed. Fluorescence changing methods of detecting nucleic acids have several advantages, including that the unbound probe molecules can easily be distinguished from those bound to the desired target without the need of a washing step, and the methods can be used either in solution or on solid supports. Most importantly, they could be applied in intact cells because no washing is needed. Moreover, fluorescence changing methods that rely on simple intensity variation by changes in quenching have the further advantage of freeing more spectral ranges so that simultaneous probing of multiple analytes can be achieved.
The most well-developed quenching-based approach to nucleic acid detection is that of “molecular beacons,” which consist of hairpin-forming DNAs labeled in the stem with a fluorophore and a quencher. The hairpin-forming DNA probe binds to a complementary sequence resulting in the hairpin opening and the quencher moving away from the fluorophore. These molecular beacons can be used in solution or in solid-supported approaches. However, this method is limited because the fluorescence change clearly depends on solution conditions, e.g. temperature, ionic strength, and thus conditions must be monitored carefully. Another disadvantage is that molecular beacon method is not as sequence selective as other DNA-sensing methods such as enzymatic approaches or some non-enzymatic autoligation methods. When the molecular beacon approach was recently used to image RNA in live mammalian cells, the results were disputed because these probes can give false positives by being degraded or by binding a protein instead of RNA. In fact, one beacon is known that binds a specific protein and lights up Fang, X.; et al., Anal. Chem. 72: 3280-3285 (2000)). There are many DNA- and RNA-binding proteins in a cell, so false positives are likely due to nonspecific binding of the probes.
The use of multicolored hairpin-shaped oligonucleotide probes (molecular beacons) was suggested for discriminating alleles (S. Tyagi et al., Nature Biotechnology 16: 49-53, 1998). The hairpin probes were reported as having significantly enhanced specificity as compared to linear probes. However, the reported specificity is not as high as phosphorothioate-iodide autoligation probes. As described above, such beacons suffer from false positives by binding proteins.
In early work, the preparation of nucleoside S-alkyl phosphorothioates was offered in 1969 (A. F. Cook, J. Am. Chem. Soc. 92(1): 190-195, 1969). The phosphorothioate group has a higher nucleophilicity than does the oxygen analog. Reactions with various halogen compounds was described. Intermolecular nucleophilic reactions of thymidine 3′-phosphorothioates were suggested in 1971 (S. Chladek and J. Nagyvary, J. Am. Chem. Soc. 94(6): 2079-2085, 1971). Dinucleotides and trinucleotides containing P-S-C 5′ linkages were formed. Those reactions were not performed with oligonucleotides, nor were they used in the detection of DNAs or RNAs, nor did they contemplate fluorescent labels or quenchers.
U.S. Pat. Nos. 5,476,925, 5,646,260, 5,648,480 and 5,932,718 suggested the preparation and use of oligonucleotides having particular internucleoside linkages. The oligonucleotides are purported to have improved hybridization properties as compared to conventional oligonucleotides.
Coupling of oligonucleotides via displacement of a 5′-O-tosyl group by a 3′-phosphorothioate was suggested by Herrlein et al. (J. Am. Chem. Soc. 117: 10151-10152, 1995). The approach was illustrated by three different systems: ligation of a nicked dumbbell oligonucleotide, cyclization of a conjugate possessing a short oligonucleotide overlap at the juncture site, and closure of a cap at the end of a duplex. Herrlein et al. do not contemplate iodides or other leaving groups such as quenching leaving groups, and they do not use the coupling to detect DNA or RNA sequences.
The displacement of an α-haloacyl group by a phosphorothioate group is suggested as a non-enzymatic method of joining two oligonucleotides by U.S. Pat. No. 5,476,930. The two oligonucleotides are brought into close proximity by binding at adjacent positions on a target polynucleotide. No quenching leaving groups were suggested.
The use of 5′-iodonucleosides was shown to allow efficient non-enzymatic ligation of single-stranded and duplex DNAs (Y. Xu and E. T. Kool, Tetrahedron Lett. 38(32): 5595-5598, 1997). An iodothymidine phosphoramidite enabled the placement of a 5′-iodide into oligonucleotides. Quenching leaving groups were not suggested in this publication.
There still exists a need for a simple method for detection and imaging of nucleic acids that is fast and accurate. Additionally, methods that are not dependent on washing away of unbound probes would be desirable, especially methods that can be used in living cells and that have specificity for as little as single nucleotide differences in sequence.
Recently, Sando and Kool published on the internet a description of the use of a quencher as a leaving group in solution and on solid phase beads (J. Am. Chem. Soc. 124(10): 2096-2097, 2002; placed on the internet on Feb. 13, 2002).